Pancreatic carboxyl ester : a circulating that modifies normal and oxidized in vitro.

R Shamir, … , D W Morel, E A Fisher

J Clin Invest. 1996;97(7):1696-1704. https://doi.org/10.1172/JCI118596.

Research Article

Pancreatic carboxyl ester lipase (CEL) hydrolyzes cholesteryl esters (CE), (TG), and lysophospholipids, with CE and TG hydrolysis stimulated by cholate. Originally thought to be confined to the gastrointestinal system, CEL has been reported in the plasma of humans and other mammals, implying its potential in vivo to modify associated with LDL, HDL (CE, TG), and oxidized LDL (lysophosphatidylcholine, lysoPC). We measured the concentration of CEL in human plasma as 1.2+/-0.5 ng/ml (in the range reported for lipase). Human LDL and HDL3 reconstituted with radiolabeled lipids were incubated with purified porcine CEL without or with cholate (10 or 100 microM, concentrations achievable in systemic or portal plasma, respectively). Using a saturating concentration of lipoprotein-associated CE (4 microM), with increasing cholate concentration there was an increase in the hydrolysis of LDL- and HDL3-CE; at 100 microM cholate, the present hydrolysis per hour was 32+/-2 and 1.6+/-0.1, respectively, indicating that CEL interaction varied with lipoprotein class. HDL3-TG hydrolysis was also observed, but was only approximately 5-10% of that for HDL3-CE at either 10 or 100 microM cholate. Oxidized LDL (OxLDL) is enriched with lysoPC, a proatherogenic compound. After a 4-h incubation with CEL, the lysoPC content of OxLDL was depleted 57%. Colocalization of CEL in the vicinity of OxLDL formation was supported by demonstrating in human aortic homogenate a cholate-stimulated […]

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Pancreatic Carboxyl Ester Lipase: A Circulating Enzyme That Modifies Normal and Oxidized Lipoproteins In Vitro

Raanan Shamir,*‡ William J. Johnson,* Kelly Morlock-Fitzpatrick,* Reza Zolfaghari,* Ling Li,§ Eric Mas,ʈ Dominique Lombardo,ʈ Diane W. Morel,* and Edward A. Fisher*§ *Department of Biochemistry, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 19129; ‡Division of Gastroenterology and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104; §Cardiovascular Institute, Mount Sinai School of Medicine, New York 10029; and ʈINSERM U260, Faculté de Médicine, 13385 Marseille, France

Abstract Introduction

Pancreatic carboxyl ester lipase (CEL) hydrolyzes choles- Modification by , such as hepatic lipase (1), lipoprotein teryl esters (CE), triglycerides (TG), and lysophospholipids, lipase (2), and A2 (3), alters the composition, with CE and TG hydrolysis stimulated by cholate. Origi- structure, and function of lipoproteins. Neglected in this re- nally thought to be confined to the gastrointestinal system, gard is the pancreatic carboxyl ester lipase (CEL,1 sometimes CEL has been reported in the plasma of humans and other referred to as bile salt–dependent cholesteryl ester ), mammals, implying its potential in vivo to modify lipids as- whose primary function is thought to be hydrolysis of dietary sociated with LDL, HDL (CE, TG), and oxidized LDL (lyso- esters in the intestinal lumen (for a recent review, see ref- phosphatidylcholine, lysoPC). We measured the concentra- erence 4). tion of CEL in human plasma as 1.2Ϯ0.5 ng/ml (in the One reason for this neglect may be the lack of appreciation range reported for ). Human LDL and that CEL activity is expressed outside of the pancreas in a HDL3 reconstituted with radiolabeled lipids were incubated number of mammalian species. For example, there are reports with purified porcine CEL without or with cholate (10 or of CEL activity and/or mRNA in rat liver (e.g., references 100 ␮M, concentrations achievable in systemic or portal 5–7), human breast (8), human hepatocarcinoma HepG2 cells plasma, respectively). Using a saturating concentration of (9), human placenta (10), rat (11) and rabbit heart (12), and rat lipoprotein-associated CE (4 ␮M), with increasing cholate and rabbit aorta (13, 14). Particularly intriguing is the evidence concentration there was an increase in the hydrolysis of that CEL is found in the blood plasma of a number of mam- LDL- and HDL3-CE; at 100 ␮M cholate, the percent hydro- mals, including humans (15), rats (11), dogs, and goats (16). lysis per hour was 32Ϯ2 and 1.6Ϯ0.1, respectively, indicat- Implied, then, is that lipoproteins in the circulation or intersti- ing that CEL interaction varied with lipoprotein class. tium could be potential targets for CEL and also that circulat- ف HDL3-TG hydrolysis was also observed, but was only 5– ing CEL, which has a heparin binding site (17), may become 10% of that for HDL3-CE at either 10 or 100 ␮M cholate. associated with a variety of tissues, independent of their capac- Oxidized LDL (OxLDL) is enriched with lysoPC, a pro- ity to synthesize the enzyme, by associating with cell-surface atherogenic compound. After a 4-h incubation with CEL, proteoglycans. The consequences of CEL-mediated lipopro- the lysoPC content of OxLDL was depleted 57%. Colocal- tein modification are suggested by recent reports that (a) the ization of CEL in the vicinity of OxLDL formation was sup- treatment in vitro of LDL by nonmammalian cholesteryl es- ported by demonstrating in human aortic homogenate a terases depletes LDL of core cholesteryl esters (18) and leads cholate-stimulated cholesteryl ester hydrolytic activity in- to increased net transfer of LDL- to cells (19), and hibited by anti–human CEL IgG. We conclude that CEL (b) CEL secreted by HepG2 cells increases the cellular uptake has the capability to modify normal human LDL and HDL of cholesteryl esters from HDL (20). composition and structure and to reduce the atherogenicity In addition to its activity against the dietary lipids choles- of OxLDL by decreasing its lysoPC content. (J. Clin. Invest. teryl ester (CE), (TG), and retinyl ester, CEL has 1996. 97:1696–1704.) Key words: low density lipoprotein • significant activity against lyosophospholipids and, in fact, was high density lipoprotein • oxidized low density lipoprotein • originally cloned as the pancreatic (21). aorta • lysophosphatidyl choline Thus, it is plausible that CEL may modify not only the core CE or TG of a normal lipoprotein, but also the lysophosphatidyl- choline (lysoPC) of oxidized LDL (OxLDL). Given the major roles attributed to lysoPC in the promotion of atherosclerotic disease (such as enhanced monocyte chemotaxis [22], mito- genic stimulation of macrophages [23], and inhibition of endo- Address correspondence to Dr. Edward A. Fisher, Mount Sinai Car- thelium-dependent arterial relaxation [24]) and the reports diovascular Institute, The Rockefeller University, Box 4, 1230 York that CEL activity was found in rat and rabbit aortae (13, 14), Ave., New York, NY 10021. Phone: 212-327-7452; FAX: 212-327- CEL could serve as a protective factor in vascular tissue 7454; E-mail: [email protected] Received for publication 21 September 1995 and accepted in re- vised form 16 January 1996. 1. Abbreviations used in this paper: CE, cholesteryl ester; CEL, car-

J. Clin. Invest. boxyl ester lipase; HDL3, HDL, subclass 3; LpL, lipoprotein lipase; © The American Society for Clinical Investigation, Inc. lysoPC, lysophosphatidylcholine; OxLDL, oxidized LDL; PC, phos- 0021-9738/96/03/1696/09 $2.00 phatidylcholine; TBARS, thiobarbituric acid–reactive substances; Volume 97, Number 7, April 1996, 1696–1704 TG, triglyceride or triacylglycerol.

Lipoprotein Modification by Pancreatic Carboxyl Ester Lipase 1696

against the adverse effects of OxLDL. Supporting this hypoth- phoresis of native and reconstituted lipoproteins and gel staining by esis are the recent reports that the treatment of OxLDL with a Sudan black were performed using Beckman Paragon Lipo Gel mate- bacterial lysophospholipase reduced the antagonism by rials. lysoPC of the endothelial-dependent relaxation response to Preparation and characterization of oxidized human LDL acetylcholine of aortic rings (24, 25). Since oxidation of LDL is thought to occur not in plasma but within arterial tissue (26), Human LDL, isolated as above, was oxidized by a 7-h incubation with Cu2+ (5 ␮M above EDTA concentration [37]). Butylated hy- aortic CEL activity would be in the appropriate location to droxytoluene (20 ␮M) was then added to stop any further oxidation. serve as a protective factor. The cholesterol content of LDL and OxLDL was determined enzy- In the studies summarized in this report, we have demon- matically by the Sigma cholesterol diagnostic kit No. 352. Protein strated: (a) the ability of CEL to hydrolyze the core lipids of content was assayed using a modification of the Lowry technique human LDL and HDL using bile salt concentrations that can (35). The cholesterol contents of LDL and OxLDL were 1.4 mg and occur outside of the intestinal lumen in healthy individuals; (b) 1.2 mg/mg protein, respectively. The protein concentrations of LDL the ability of CEL to reduce the lysoPC content of human Ox- and OxLDL were 1.4 and 1.3 mg/ml, respectively. The extent of lipid LDL; (c) the presence of CEL in human plasma; and (d) the peroxidation was estimated as thiobarbituric acid–reactive substances presence of an activity in human aortic homogenate with enzy- (TBARS) as described previously (38). Briefly, LDL or OxLDL (50 ␮ matic and immunological properties indistinguishable from g protein of either) and various amounts of standard (1,1,3,3-tet- ramethoxypropane) were mixed with 1 ml of 25% trichloroacetic acid CEL. The results strongly suggest that CEL plays a wider role and 1 ml of 1% 2-thiobarbituric acid containing 5 ␮M CuSO4. The in lipid and lipoprotein metabolism than previously appreci- mixture (final volume of 0.4 ml) was incubated at 95ЊC for 45 min and ated both in terms of enzymatic targets and tissue distribution. centrifuged at 1,500 rpm at 10ЊC for 30 min to remove any insoluble

material. The OD532 of the supernatant was determined and the Methods amount of TBARS was expressed as nanomoles of malondialdehyde equivalents per milliliter of reaction mixture. All chemicals were the highest grade commercially available and pur- chased from Sigma Chemical Co. (St. Louis, MO), unless otherwise Human tissue collection and preparation noted. Cholesteryl [1-14C]oleate (supplied with specific activity of Male and female aortic arch samples were obtained at autopsy and 14 53.4 mCi/mmol), [carboxyl- C]triolein (112 mCi/mmol), and [palmi- frozen immediately in liquid N2 and then stored at Ϫ70ЊC until ana- toyl-114C]lysopalmitoyl phosphatidylcholine (56.7 mCi/mmol) were lyzed. Under the Institutional Review Board guidelines autopsy sam- purchased from New England Nuclear (Boston, MA). [1␣,2␣(n)- ples are collected so that neither the identity nor cause of death of 3H]cholesteryl oleate (specific activity of 49.6 Ci/mmol) was pur- subjects was revealed to us. To prepare samples for enzymatic analy- chased from Amersham (Arlington Heights, IL). Porcine pancreatic sis frozen tissues were thawed on ice. Adherent vascular and mem- CEL was purified as reported previously (27). Reagents for lipopro- branous materials were dissected away and the remaining tissue dis- tein electrophoresis were obtained from Beckman (Fullerton, CA). persed at 4ЊC in 3 vol of 0.25 M sucrose at full speed for 1 min in a The procedures for obtaining human blood samples have been homogenizer (Tekmar Co., Cincinnati, OH). Aliquots were stored approved by the Committee for Protection of Human Subjects at the frozen at Ϫ70ЊC until enzymatic assays were performed (see below). Medical College of Pennsylvania and the donors have signed in- formed consent forms. Assay of the hydrolysis of CE, TG, or lysoPC All activity determinations were done in duplicate with a variability Reconstitution of human lipoproteins with radiolabeled lipids coefficient Ͻ 10%. Lipoprotein fractions were isolated from the plasma of normal hu- CE and TG. For reconstituted lipoproteins, the hydrolysis of CE man volunteers using sequential density gradient centrifugation (28, or TG was determined based on radiometric procedures described

29). HDL3 was delipidated with ethanol/diethyl ether (3:2 vol/vol) at previously (11, 39). For substrates dispersed in ethanol, the reaction

0ЊC (30) and the apoproteins reserved for the reconstitution step (see mixture had a final volume of 0.2 ml; for substrates in LDL or HDL3, below). The ethanol/diethyl ether lipid extracts were dried by rotary the final volume was 1 ml. The final concentration of Tris-maleate evaporation and stored in 2:1 chloroform/methanol. To incorporate (pH 7.0) was 50 mM, and sodium cholate was either 0, 10, or 100 ␮M. 14 14 cholesteryl [ C]oleate or [ C]triolein into HDL3, the labeled com- Reactions were initiated by addition of purified CEL and labeled pounds were added directly to the lipid extract, which was then re- substrate (to the concentrations given in Results) and incubated at

constituted with HDL3 apoproteins by sonication (31). The reconsti- 37ЊC for the lengths of time also given in Results. The released 14 ف tuted HDL3 had a final specific activity of 0.022 ␮Ci of cholesteryl [ C]oleate was extracted as described previously (11) and quanti- ␮Ci of [carboxyl-14C]triolein per tated by scintillation counting. Results are typically expressed in units 7 ف 1-14C]oleate per ␮mol CE or] ␮mol TG. of 1 pmol fatty acid released/h/␮g CEL. To examine the effect of [1␣,2␣(n)-3H]cholesteryl oleate was incorporated into LDL by CEL on the hydrolysis of CE of native LDL a similar protocol was the potato-starch method of Krieger et al. (32). The final specific ac- performed, but the reaction volume was 5 ml and cholesterol (total, -␮Ci of [1␣,2␣(n)-3H]cholesteryl oleate per ␮mol free, ester) was assayed as in the above section, Analysis of reconsti 0.10 ف tivity was LDL-CE. tuted and native human lipoproteins. For analysis of aortic homogenates, 50 ␮l of an appropriately di- Analysis of reconstituted and native human lipoproteins luted homogenate was used as the enzyme source. The final reaction For reconstituted lipoproteins, lipids were extracted by the procedure volume was 0.2 ml and the concentration of Tris-maleate (pH 7.0) of Bligh and Dyer (33). Phospholipid was determined by the method was 50 mM. Cholate or deoxycholate was added at a concentration of Sokoloff and Rothblat (34). Protein was assayed by a modification ranging from 0 to 100 mM as indicated in Results. The reactions were of the Lowry technique (35). Cholesterol (free and total) was deter- initiated by addition of 0.01 ml ethanol containing 2 nmol of choles- mined by gas-liquid chromatography using choelsteryl methyl ether teryl [1-14C]oleate with a specific activity of 25 ␮Ci/␮mol. The tubes as an internal standard (36). Esterified cholesterol was calculated by were incubated at 37ЊC for 30 min. The released [14C]oleate was ex- subtracting free cholesterol from total cholesterol. TG was deter- tracted and quantitated as above. Results are expressed in units of 1 mined by the Sigma triglyceride diagnostic kit, using glycerol as a nmol [14C]oleate released/h/gram of tissue. standard. In some experiments, the cholesterol content (total, free, To investigate the inhibition of aortic homogenate CEL activity ester) of native LDL was determined as above. Agarose electro- by a rabbit IgG directed against human CEL, the following experi-

Lipoprotein Modification by Pancreatic Carboxyl Ester Lipase 1697

ment was performed. Aortic homogenate was preincubated overnight PBS/Tween, 100 ␮l of p-nitrophenyl phosphate (1 mg/ml in 0.2 M at 4ЊC with 5 ␮g of either rabbit anti–human CEL (40, 41) or rabbit Tris/HCl, pH 8.5, 1 mM CaCl2, and 1 mM MgCl2) was added to each preimmune IgG. The samples were then assayed for CEL activity as well and incubated for 1 h at 37ЊC. The degree of color development described above. was determined by reading the plate in an MR 5000 spectrophotome- LysoPC. Lysophospholipase activity was measured using the ter (Dynatech Laboratories Inc., Chantilly, VA) at 405 nm and com- method described by Vanden Bosch et al. (42). Substrate and either paring the serum values with the standard curve based on the assay buffered enzyme (porcine pancreatic CEL or bacterial 2-lysophos- results for serially diluted samples of the pure human CEL. phatidylcholine acylhydrolase [EC 3.1.1.5; Sigma]) or buffer alone Data analysis (potassium phosphate 0.1 M) were diluted with dH2O to a final vol- ume of 0.4 ml and a final potassium phosphate concentration of 200 Unless otherwise indicated, data are expressed as mean valuesϮSD mM. In some cases, to determine the presence of endogenous lyso- and statistical comparisons made by the Student’s t test or ANOVA. phospholipase activities of LDL and OxLDL, these lipoproteins were The software program InStat (GraphPAD Software for Science, San substituted for the enzyme source. Diego, CA) was used for testing statistical significance. The reaction was started by adding 200 nmol (0.1 ml of a 2 mM 14 aqueous solution) of the substrate [palmitoyl-1- C]lysopalmitoyl Results phosphatidylcholine with specific activity of 26.1 ϫ 10Ϫ3 ␮Ci/␮mol. After 10 min at 37ЊC, the reaction was stopped with 2.5 ml of modi- CEL is a circulating lipase in humans fied Dole’s solution (43). Approximately 100 mg of silica gel (100–200 mesh) was added, followed by 1.5 ml of heptane and 1.5 ml of water. CEL activity or mass has been reported in the plasma of rats After each addition, the tube was vortexed for 15 s. 1 ml of the result- (11), dogs, goats (16), and humans (15). We were interested in ing upper phase, which contained the labeled fatty acid released from confirming that CEL is a circulating enzyme in humans. lysoPC by hydrolysis, was taken for scintillation counting. Results are Plasma CEL mass was measured in six normal volunteers by a expressed in nanomoles of fatty acid released per minute per milli- quantitative ELISA assay (15). The average concentration was gram of protein. Hydrolysis of lysoPC at a final concentration of 80 1.2Ϯ0.5 ng/ml (range 0.6–2.0), consistent with literature values ␮M (similar to the concentration of OxLDL-associated lysoPC in the (15) and in the range reported for lipoprotein lipase (LpL) experiments described below) was also assayed, but with 4 ␮g/ml of (44). CEL and for incubation times up to 240 min. The percent hydrolysis at each time point was calculated from the scintillation counting data. The effects of CEL on normal lipoproteins In pilot experiments, the potential for CEL to act as a phospholi- Properties of reconstituted human HDL3 and LDL. The recon- pase was investigated using both radiolabeled phosphatidylcholine stitution of normal human HDL3 and LDL included radiola- (PC) and HDL reconstituted with radiolabeled PC. No activity above beled CE. Since human HDL also contains small amounts of background was detected in either case. 14 TG, some HDL3 was also reconstituted with [ C]TG as the Effect of CEL on the lysophospholipid content of OxLDL only labeled core lipid. As shown in Table I, the chemical com- -mg protein/ml positions of the reconstituted lipoproteins resembled the re 0.5 ف Human lipoproteins (LDL, OxLDL) containing were incubated with either 4 ␮g CEL protein/ml or 1 ␮g/ml bacterial ported values for native human lipoproteins (45). In addition, 2-lysophosphatidylcholine acylhydrolase or buffer alone (potassium we tested whether the cholate concentrations used in the ex- phosphate, 200 mM final concentration) for 4 h at 37ЊC. At time periments described below would significantly affect the physi- points (0, 1, 2, and 4 h) the reaction was stopped with 1 ml methanol cal properties of the reconstituted lipoproteins. The agarose and lipids were extracted using the procedure of Bligh and Dyer (33). gel electrophoretic mobilities of reconstituted LDL and HDL3 The phospholipids were separated by TLC on silica gel G (250 ␮m as were not affected by treatment with 10 or 100 ␮M cholate TM Uniplate ; Analtech Inc., Newark, DE) with two solvent systems (data not shown), indicating that the bile acid did not cause used in the same dimension: chloroform/methanol/water, 65:35:6 major disruptions of the structure of these lipoproteins. (vol/vol/vol) and chloroform/acetone/methanol/acetic acid/water, 6:8: Characterization of the action of CEL on the CE of HDL . 2:2:1. After visualization with iodine vapor, PC and lysoPC bands 3 were scraped from the plates and assayed for inorganic phosphorus Since CE is the major core lipid in HDL, and this lipoprotein is 14 content according to the method of Sokoloff and Rothblat (34). Each relatively simple to reconstitute, we used [ C]CE-labeled TLC plate had lanes containing unlabeled and labeled standards to HDL3 to establish appropriate reaction conditions for examin- localize the desired bands in the sample lanes. Quantitative recovery ing lipoprotein modification by CEL. Previous studies of lipo- of lysophospholipids was confirmed in some experiments by adding proteins modified by cholesteryl have typically used trace amounts of lysoPC to samples before lipid extraction and TLC in the milligrams per milliliter range (e.g., references separation. Detection of circulating CEL in human serum Table I. Chemical Compositions of Human HDL and LDL The amount of CEL in human serum samples was determined by 3 Reconstituted with Labeled Core Lipids ELISA as described previously (15). Briefly, wells of immunoplates (Nunc, Roskilde, Denmark) were coated with a 10 ␮g/ml solution (0.1 LDL HDL3 HDL3 M carbonate buffer, pH 9.5) of anti–human CEL polyclonal antibod- [3H]CE [14C]CE [14C]Triolein ies rased in rabbit (40, 41). After overnight incubation at 4ЊC, wells were washed three times for 30 min at 37ЊC with 0.05% Tween 20 in Protein 27.4Ϯ2.1 48.0Ϯ0.07 41.0Ϯ0.05 PBS (PBS/Tween). Then, 100-␮l portions of each sample (either hu- Phospholipid 29.8Ϯ1.6 32.2Ϯ0.04 32.4Ϯ0.90 man serum or pure human CEL standard), appropriately diluted in CE 23.7Ϯ1.4 15.4Ϯ1.20 20.3Ϯ1.53 PBS/Tween, were added to the wells and incubated for 30 min at Cholesterol 15.3Ϯ1.5 2.90Ϯ0.05 3.78Ϯ0.11 37ЊC. After three washes with PBS/Tween, 100 ␮l of biotin-labeled TG 3.84Ϯ0.63 1.57Ϯ0.03 2.52Ϯ0.18 rabbit polyclonal antibodies to human CEL (2 ␮g/ml) was added to each well and incubated for 2 h at 37ЊC. After three washes with PBS/ Tween, dilute alkaline –avidin conjugates were added The chemical composition was determined in triplicate and is given as (100 ␮l/well) and incubated for 2 h at 37ЊC. After three washes with percentageϮSD of total weight.

1698 Shamir et al. 18 and 19). However, given the similar concentrations in hu- Table II. Hydrolysis by Pancreatic CEL of CE and TG man plasma of LpL and CEL noted above, we decided to use a Dispersed in Ethanol or Associated with Lipoproteins concentration in the micrograms per milliliter range, similar to (pmol/h/␮g CEL Protein ϮSD) those used in studies of LpL-lipoprotein interactions in vitro No cholate 10 ␮M cholate 100 ␮M cholate (e.g., references 46 and 47). The next choice was the range of cholate concentration. The concentration of bile acid in human Ethanol fold higher in CE ND 110Ϯ9 4049Ϯ490-10 ف plasma is in the range of 10 ␮M (48) and is postprandial portal plasma (49). Thus, cholate concentrations TG ND 3980Ϯ1044 8518Ϯ2168 of 0, 10, and 100 ␮M were used. LDL With the chosen CEL and cholate concentrations, the de- CE 27Ϯ1 185Ϯ16 5818Ϯ345 pendences of HDL3-CE hydrolysis on incubation time and substrate concentration were examined. Independent of cho- HDL3 late concentration, the hydrolysis of CE did not approach a CE 4Ϯ120Ϯ223Ϯ1 plateau until after 60 min (Fig. 1 A), but clearly saturated at a TG 0.3Ϯ0.0 1.2Ϯ0.3 2.5Ϯ0.2 CE concentration of 4 ␮M (Fig. 1 B). These parameters of in- cubation time (60 min) and lipid concentration (4 ␮M) were CEL activity was assayed at 37°C for 60 min using (a) CE (4 ␮M) dis- maintained in subsequent testing of the effects of CEL on the persed in ethanol, (b) TG (4 ␮M) dispersed in ethanol, (c) reconstituted rates of hydrolysis of HDL3-TG and LDL-CE in order to LDL (4 ␮M CE), (d) reconstituted HDL3 (4 ␮M CE), (e) reconstituted make comparisons among results obtained under identical HDL3 (4 ␮M TG). Purified CEL was used at 4 ␮g/ml except for sub- conditions. strates a and c when the cholate concentration was 100 ␮M; in these The effects of substrate presentation on CEL-mediated hy- cases, the CEL concentration was 0.25 ␮g/ml. ND, Activity above back- drolysis of CE and TG. Using the conditions defined above, ground not detected. CE was presented to CEL in one of the following forms: (a) dispersed in EtOH, (b) associated with HDL3, or (c) associ- ated with LDL. In addition, TG was presented in either form a deplete the CE content of LDL to a greater extent than that of or b. HDL3, given not only the increased fractional hydrolysis of The results are summarized in Table II. Note that for all LDL-CE, but also that the CE core of LDL is much larger forms of substrate presentation, 10 ␮M cholate significantly than that of HDL3. In Table III are summarized experiments stimulated hydrolysis in comparison to the no cholate control. in which the fractional hydrolysis per hour of LDL-CE and

In all but one case (HDL3-CE), increasing the cholate concen- HDL3-CE by CEL was measured. As shown, there was indeed tration from 10 to 100 ␮M further stimulated hydrolytic activ- an appreciable percentage of LDL-CE hydrolyzed over time ity. It is also interesting to note that for EtOH-CE or LDL-CE, at either cholate concentration. That hydrolysis of LDL-CE by at either concentration of cholate, the hydrolysis was compara- CEL and its stimulation by cholate were not artifacts of the li- ble and greatly exceeded that observed for HDL3-CE. Simi- poprotein reconstitution procedure was supported by finding larly, the hydrolysis of EtOH-TG was significantly greater similar results with native LDL studied under the same condi- than that of HDL3-TG. tions (4 ␮M LDL-CE and 0.25 ␮g/ml CEL): at 0 and 100 ␮M These results suggested that modest amounts of CEL could cholate, there were reductions in CE mass of 1.3% (Ϯ0.7, n ϭ

Figure 1. Dependence of CEL- mediated hydrolysis on incuba- tion time and substrate concen- tration using reconstituted

HDL3. (A) CEL activity was measured at 37ЊC as described in Methods using reconstituted

HDL3 (4 ␮M CE). With 0 or 10 ␮M cholate, 4 ␮g/ml CEL was used. With 100 ␮M cholate, the concentration of CEL was 0.25 ␮g/ml. The amount of [14C]ole- ate released was determined as described in Methods and ex- pressed as picomoles per micro- gram of CEL. (B) CEL activity was measured as above, except the reaction time was 1 h and the amount of reconstituted

HDL3 varied to supply the indi- cated CE concentrations. ᮀ, no cholate; ᭡, 10 ␮M cholate; ᭺, 100 ␮M cholate; *differs from 0 cholate at P Ͻ 0.01; ** differs at P Ͻ 0.001.

Lipoprotein Modification by Pancreatic Carboxyl Ester Lipase 1699 Table III. Hydrolysis in 1 h by CEL of LDL-[3H]cholesteryl 14 14 Oleate (CO), HDL3-[ C] CO, or HDL3-[ C]triolein (TO)

Percent hydrolysis

3 14 14 [Cholate] LDL-[ H]CO HDL3-[ C]CO HDL3-[ C]TO

0 2.1Ϯ0.1 0.3Ϯ0.03 0.03Ϯ0.007 10 ␮M 14.6Ϯ1.3 1.61Ϯ0.18 0.06Ϯ0.01 100 ␮M 32.0Ϯ1.9 1.64Ϯ0.14 0.10Ϯ0.03

In a set of reactions separate from those summarized in Table II, hydroly- sis by purified CEL was assayed using (a) reconstituted LDL (4 ␮M

CE), (b) reconstituted HDL3 (4 ␮M CE), (c) reconstituted HDL3 (4 ␮M TG). Purified CEL was used at 4 ␮g/ml except for substrate with cholate at a concentration of 100 ␮M; in this case, the CEL concentration was 0.25 ␮g/ml.

Figure 2. Percent hydrolysis by CEL of lysoPC. Lysophospholipase activity was measured as described in Methods. The concentrations of 4) and 35.0% (Ϯ5.8, n ϭ 4), respectively. lysoPC and CEL were 80 ␮M and 4 ␮g/ml, respectively. The reactions In contrast to the LDL results, the percentage of the were stopped at the indicated times, then the released fatty acids HDL3-CE hydrolyzed under comparable conditions was rela- were extracted and quantitated as described in Methods. Percent hy- tively minor. Nonetheless, given the long half-life of HDL drolysis was determined as 100 ϫ (extracted cpm/total cpm) and is .d) in the human circulation (for review see reference 50), presented as meanϮSD, n ϭ 3 3 ف) the extent of hydrolysis of HDL-CE in vivo may be underesti- mated by the results in vitro. creatic CEL was present in aortic homogenate (13, 14). To The effects of CEL on OxLDL pursue whether there is a similar activity in human aorta, tis- Characterization of lipoproteins. TBARs for LDL and Ox- sue samples were obtained at autopsy. There was significant LDL were 4.3Ϯ0.5 (n ϭ 4) and 52.9Ϯ0.9 (n ϭ 4) nmol of mal- activity present in 10/11 males (for n ϭ 11: 28.9Ϯ10.3 nmol CE ondialdehyde equivalents/mg protein, respectively. As ex- hydrolyzed/h/gram of tissue, meanϮSE; range of 0–122) and pected, compared with LDL, OxLDL demonstrated increased 8/10 females (for n ϭ 10: 11.1Ϯ4.7; range of 0–46.4). Although anodic electrophoretic mobility (data not shown), reflecting a there was a higher mean level in males, because of the interin- surface more negatively charged. In the absence of lipases, the dividual variability the gender difference was not statistically PC and lysoPC contents of LDL and OxLDL did not change significant, unless the analysis was restricted to 10 males and 8 during 4 h of incubation. Lysophospholipase activities of LDL and CEL. Lysophos- pholipase activity was determined using the assay described earlier (see Methods) under conditions of a saturating concen- tration of substrate. The endogenous activity of native or Ox- LDL was not different from background (data not shown). The activity of the porcine pancreatic CEL was 6,740Ϯ205 nmol of lysoPC hydrolyzed/min/mg protein (n ϭ 4). As shown in Fig. 2, when CEL (4 ␮g/ml) was incubated with lysoPC in aqueous solution using a concentration similar to that of lysoPC associated with OxLDL, hydrolysis was 6.1%Ϯ0.8 and 47.2%Ϯ4.7 after 15 min and 4 h, respectively. Effect of CEL on lysoPC content of OxLDL. The lysoPC content of OxLDL was determined after 4 h of incubation with and without CEL. As shown in Fig. 3, the content of lysoPC was unchanged by incubation for 4 h without CEL, but after 4 h with CEL, the lysoPC content was significantly (P Ͻ 0.01) reduced to 43% of the initial value. Note that a comparable re- duction was also obtained using the bacterial lysophospholi- pase () used in previous studies (24). Thus, the lysoPC associated with OxLDL is available to the lyso- phospholipase activity of CEL. Figure 3. Effect of CEL on lysoPC content of OxLDL. OxLDL was CEL activity in human aorta. Because oxidation of LDL is incubated with either 4 ␮g/ml of CEL, 1 ␮g/ml of bacterial 2-lyso- phosphatidylcholine acylhydrolase, or buffer alone for 4 h at 37ЊC. At thought to occur not in plasma but after the entry of LDL into the indicated times, the reaction was stopped and the lysoPC content the subintimal space of arterial tissue (26) the presence of CEL remaining was determined as described in Methods. Results are given where OxLDL is generated and exerts it deleterious effects as meanϮSD, n ϭ 6. ᭹, bacterial 2-lysophosphatidylcholine acylhy- would be particularly significant. It has been reported previ- drolase; ᭡, CEL; and ᭿, buffer alone; *differs from buffer alone at ously that in rat and rabbit an activity resembling that of pan- P Ͻ 0.001.

1700 Shamir et al. shown in Fig. 5, preincubation of human aortic homogenate with a small amount (5 ␮g) of rabbit anti-human CEL anti- body significantly (P Ͻ 0.01) reduced the hydrolysis of CE compared with treatment with an equivalent amount of rabbit nonimmune serum.

Discussion

Besides its role in the digestive process (for review see refer- ence 4), systemic actions of CEL must be considered, given that it circulates in a number a mammalian species (11, 16), in- cluding humans (15). Since other lipases that circulate have been shown to modify normal human HDL and LDL (e.g., LpL [1] and hepatic lipase [2]), it was natural to investigate whether the lipids on these lipoproteins could be hydrolyzed by CEL. However, one point we needed to take into account was that the millimolar concentrations of bile acid required for Figure 4. Cholesteryl ester hydrolase activity stimulated by trihy- droxy bile salt in human aortic homogenate. CEL hydrolysis of CE in maximal hydrolysis of CE and TG in the intestine are not the presence of trihydroxy (cholate) or dihydroxy (deoxycholate) bile found in either plasma or interstitial fluid. Instead, in systemic ف salt was determined in 50 ␮l of human aortic homogenate as de- plasma the concentration of bile acid is 10 ␮M (48) and in fold greater (49). Since-10 ف scribed in Methods. Cholate (᭿) or deoxycholate (᭡) was added at postprandial portal plasma is concentrations ranging from 0 to 100 mM. 1 unit is 1 nmol [14C]oleate both of these concentrations are well in excess of the nanomo- released/h/gram of tissue. Reactions were done in duplicate and the lar levels shown to affect the conformation of CEL (51), it was mean values are displayed. certainly possible that, in the 10–100 ␮M range physiologically obtainable outside of the intestine, there would be sufficient enzymatic activity to hydrolyze significant amounts of lipopro- females with detectable activity, in which case the male aver- tein-associated CE and TG. age (n ϭ 10: 31.8Ϯ11.0) was significantly higher (P Ͻ 0.04) As summarized in Tables II and III, this was indeed the than the female average (n ϭ 8: 13.8Ϯ5.5). case. Interestingly, the rates of hydrolysis (Table II) varied

That the homogenate activity represented bona fide CEL dramatically by mode of presentation (EtOH, HDL3, LDL). was supported by two experiments. In the first, the maximal The hydrolysis of lipoprotein-associated CE and TG in the ab- stimulation of the reaction by millimolar concentrations of tri- sence of cholate is consistent with the studies of Brockman and hydroxy (e.g., cholate) and not dihydroxy (e.g., deoxycholate) colleagues (e.g., reference 52), who have shown that some bile salts, a characteristic feature of CEL activity measured in CEL-mediated hydrolysis can occur in the absence of bile vitro (5), was determined. As shown in Fig. 4, human aortic salts, depending on the characteristics of the substrate/enzyme homogenate CEL activity increased with increasing concentra- interface. At either the 10 or 100 ␮M cholate concentration, tion of cholate, but not deoxycholate. In the second type of ex- the hydrolysis of both CE and TG associated with HDL3 was periment, the ability of an antibody raised against human CEL much lower than when the corresponding substrate was pre- (40, 41) to inhibit the homogenate activity was tested. As sented in either EtOH or associated with LDL. There are reports that with other lipases there was also dif- ferential hydrolysis of HDL and LDL lipids (e.g., references 18 and 53). For example, using a fungal cholesteryl , un- der conditions in which LDL-CE (as percentage of total cho- lesterol) went from 75 to 6%, the corresponding values for HDL-CE were 79 and 65% (18). Factors such as packing den- sity, surface charge, and apoprotein composition, all different

between HDL3 and LDL, probably affect the interaction of CEL (and the other enzymes) with lipoproteins as well as the availability to the enzymes of the substrates. In addition to the data in Tables II and III just alluded to, that such factors exert important influences on the interaction of CEL with LDL and

HDL3 is also indicated by some simple calculations (based on data in reference 54): The CE content of human LDL is 1,310 molecules/particle. Assuming a surface pool of CE of 3 mol%, the fraction of total LDL-CE that would be the functional sub- strate for the enzyme would be 20 surface molecules/1,310 to- Figure 5. Effect of anti-CEL antibody on bile salt–dependent CEL tal molecules, or 1.5%. The remaining 98.5% would be a “sub- activity in human aortic homogenate. Diluted aortic homogenate (50 ␮l) was incubated overnight at 4ЊC with 5 ␮g of either rabbit anti– strate reservoir.” A similar calculation for HDL3 is a total CE human CEL IgG or rabbit preimmune IgG. The samples were then content of 32 molecules/particle and 4 surface molecules, for a assayed for CEL activity at 50 mM cholate as described in Methods. fractional functional substrate of 4/32, or 12.5%. Thus, in con- The reactions were done in triplicate and mean valuesϮSD are dis- trast to what was observed (Table II), the initial rate of hydro- played. lysis of HDL3-CE should have been 8 (i.e., 12.5%/1.5%) times

Lipoprotein Modification by Pancreatic Carboxyl Ester Lipase 1701 greater than that of LDL-CE. The fact that LDL-CE was the In a set of studies, Henry and colleagues (24, 25) have better substrate for CEL (Tables II and III) implies that the shown that the treatment of OxLDL with a bacterial lysophos- enzyme interacts with this particle better than with HDL3. pholipase reduces not only the lysoPC content but also restores The potential consequences of the action of CEL on the the endothelial-dependent relaxation response to acetylcholine structural and functional properties of LDL and HDL are sug- of rabbit aortic rings. As shown in Fig. 3, we confirmed the abil- gested by a number of studies. First, the core content of either ity of the bacterial enzyme to reduce OxLDL lysoPC content CE or TG has been shown recently to affect the ␣-helical con- and also showed comparable effectiveness of CEL. tent of HDL–apo AI and the stabilities of both the apo AI and Since oxidation of LDL is not thought to occur in the the HDL particle itself (Sparks, D.L., W.S. Davidson, S. Lund- plasma compartment but after LDL enters the subintimal Katz, and M.C. Phillips, manuscript submitted for publica- space, to serve as a potential protective factor against lysoPC tion). Such changes could, in turn, affect the ability of HDL to the optimal location of CEL for this function would be in arte- accept cellular cholesterol or to serve as a substrate for LCAT rial tissue. The partial purification of an enzymatic activity in (55). This may be the basis for our previous finding that HDL rat and rabbit aortae (13, 14) with characteristics of pancreatic added to medium containing CEL secreted from transfected CEL encouraged us to examine human samples. That bona rat hepatoma cells promoted greater clearance of cellular cho- fide CEL was present was supported by (a) the stimulation of lesterol compared with the clearance from nonsecreting con- the cholesteryl ester hydrolase activity by only trihydroxy bile trol hepatoma cells (56). salt and not other detergent molecules (Fig. 4), a particularly (of LDL-CE by a characteristic feature of this enzyme (e.g., reference 5); and (b 3/1 ف In Aviram et al. (19), hydrolysis of bacterial enzyme resulted in a smaller particle and a 30% re- the inhibition of homogenate activity by preincubation with a duction in lipoprotein binding and degradation by the J774 specific antibody (40, 41) directed against human CEL (Fig. 5). macrophage-like cell line. The authors concluded that these The source of arterial CEL could be multiple; it could be de- results were a consequence of conformational changes in apo rived from the circulating pool or it could be endogenously B due not only to a reduction of core CE but also to a redistri- synthesized and secreted by one or more of the cell types (e.g., bution of the free cholesterol between the core and the surface leukocytes [60], smooth muscle, endothelium) present in the of the lipoprotein. In Chao et al. (18), HDL and LDL were tissue. Further studies using immunohistochemistry and in situ pretreated with trypsin, and CE was hydrolyzed by a fungal hybridization will be necessary to determine the source of aor- enzyme. Extensive hydrolysis of LDL-CE resulted in the for- tic CEL activity. It should be noted that others have also re- mation of large multilamellar free cholesterol-rich structures ported a non-CEL cholesteryl ester hydrolase activity in the resembling particles that accumulate in atherosclerotic lesions. aorta or its component cells (e.g., references 2 and 61). This ac- Hydrolysis of core TG of HDL and LDL has also been in- tivity resembles that of hormone-sensitive lipase, in that it is vestigated previously. Aviram et al. (2) treated LDL with LpL. maximally active at neutral pH and is stimulated by cAMP. In contrast to the results above, the modified LDL had a We do not think that this contributed significantly to the re- higher affinity for the LDL receptor. While the authors recon- sults in human aortic homogenate, however, since a millimolar ciled the results of their two papers by noting that predomi- concentration of cholate inhibits hormone-sensitive lipase–like nately the core of the LDL is modified by LpL, whereas both activity (reference 62 and Zolfaghari, R., and E.A. Fisher, un- the core and surface were modified by CE hydrolysis, a more published results). d) in 3 ف) likely explanation comes from later work of Williams et al. The relatively long half-lives of LDL and HDL (47) and others (e.g., references 57 and 58) showing that LpL the human circulation (for review see reference 50) and the bound to LDL can act as a structural bridge to cell-surface pro- even greater half-lives of retained lipoproteins in the subinti- teoglycans. This would be consistent with the findings of Avi- mal space of the aorta (for review see reference 63) imply that ram et al. (2) that in the mathematical analysis of the binding there may be significant modification of normal and oxidized of LpL-modified LDL to macrophages there was evidence for lipoproteins by CEL, even for the more refractory substrates an increase in binding sites. It may be that a similar phenome- on HDL. For OxLDL, CEL is likely to be a protective factor, non will be observed for CEL-modified lipoproteins, since given its ability to reduce lysoPC content. For normal lipopro- CEL has a heparin binding site that has been reported to pro- teins, it is not clear whether CEL modification would be bene- mote the interaction of the enzyme with proteoglycans (17). ficial or deleterious. For example, it could lead to the forma- This would explain the report that CEL enhanced the delivery tion of LDL particles with increased cellular binding affinity of a nonhydrolyzable core lipid of HDL (cholesteryl ether) to (resulting in increased cellular cholesterol content) or denser hepatoma cells (20). LDL particles having increased access to the aortic subintimal While many studies have focused on CEL as a triglyceride li- space. On the other hand, LDL binding could be reduced (see pase and a cholesteryl ester hydrolase, it should be noted that it reference 19) and/or efflux to HDL could be increased (56), was originally cloned as a pancreatic lysophospholipase (21) and both phenomena serving to decrease the net cellular choles- subsequently shown to be the only enzyme secreted by the pan- terol content. Nevertheless, our results clearly establish the po- creas with this activity (59). Unlike its other catalytic functions, tential for CEL to exert important effects on lipid metabolism there is no bile salt dependence under any condition for the hy- outside of the intestinal lumen and support the undertaking of drolysis of lysophospholipids. One major lysophospholipid with further studies to define the overall impact of the systemic ac- relevance to atherogenesis is lysoPC. OxLDL is enriched in tivity on the development of . lysoPC, which is thought to mediate deleterious effects in arte- rial tissue mainly by serving, for example, as a chemoattractant Acknowledgments that recruits monocytes to the subintimal space (22) or as an an- tagonist of endothelial-dependent relaxation (24). We thank Dr. Howard L. Brockman (Hormel Institute, University of Minnesota, Austin, MN) for the gift of purified CEL; Dr. Elizabeth

1702 Shamir et al. Laposata (Medical Examiner’s Office, Wilmington, DE) for provid- 23. Sakai, M., A. Miyazaki, H. Hakamata, T. Sasaki, S. Yui, M. Yamazaki, ing autopsy samples while she was at the Hospital of the University of M. Shichiri, and S. Horiuchi. 1994. Lysophosphatidylcholine plays an essential Pennsylvania; Ms. Jill Feltheimer Fisher for valuable editorial assis- role in the mitogenic effect of oxidized low density lipoprotein on murine mac- rophages. J. Biol. Chem. 269:31430–31435. tance; and our colleagues in the Department of Biochemistry, Dr. 24. Kugiyama, K., S.A. Kerns, J.D. Morrisett, R. Roberts, and P.D. Henry. Michael C. Phillips and Dr. Earl H. Harrison, for helpful discussions. 1990. Impairment of endothelium-dependent arterial relaxation by lysolecithin The technical assistance of Ms. Christine E. Ackerman and Ms. Kat- in modified low-density lipoproteins. Nature (Lond.). 344:160–162. mna Katiyar and the contribution of Dr. Camilo Rojas to the aorta- 25. 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